EP2299836B1 - Enzymatische verfahren zur geschmacksveränderung - Google Patents

Enzymatische verfahren zur geschmacksveränderung Download PDF

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EP2299836B1
EP2299836B1 EP09789870.4A EP09789870A EP2299836B1 EP 2299836 B1 EP2299836 B1 EP 2299836B1 EP 09789870 A EP09789870 A EP 09789870A EP 2299836 B1 EP2299836 B1 EP 2299836B1
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Prior art keywords
lipase
cheese
food product
lipases
fatty acids
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French (fr)
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EP2299836A2 (de
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James F. Jolly
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Amano Enzyme Inc
Amano Enzyme USA Co Ltd
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Amano Enzyme Inc
Amano Enzyme USA Co Ltd
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    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
    • A23L27/00—Spices; Flavouring agents or condiments; Artificial sweetening agents; Table salts; Dietetic salt substitutes; Preparation or treatment thereof
    • A23L27/20—Synthetic spices, flavouring agents or condiments
    • A23L27/206—Dairy flavours
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23C—DAIRY PRODUCTS, e.g. MILK, BUTTER OR CHEESE; MILK OR CHEESE SUBSTITUTES; MAKING THEREOF
    • A23C19/00—Cheese; Cheese preparations; Making thereof
    • A23C19/06—Treating cheese curd after whey separation; Products obtained thereby
    • A23C19/063—Addition of, or treatment with, enzymes or cell-free extracts of microorganisms
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
    • A23L27/00—Spices; Flavouring agents or condiments; Artificial sweetening agents; Table salts; Dietetic salt substitutes; Preparation or treatment thereof
    • A23L27/20—Synthetic spices, flavouring agents or condiments
    • A23L27/24—Synthetic spices, flavouring agents or condiments prepared by fermentation
    • A23L27/25—Dairy flavours
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
    • A23L27/00—Spices; Flavouring agents or condiments; Artificial sweetening agents; Table salts; Dietetic salt substitutes; Preparation or treatment thereof
    • A23L27/88—Taste or flavour enhancing agents
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y301/00—Hydrolases acting on ester bonds (3.1)
    • C12Y301/01—Carboxylic ester hydrolases (3.1.1)
    • C12Y301/01003—Triacylglycerol lipase (3.1.1.3)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y302/00—Hydrolases acting on glycosyl compounds, i.e. glycosylases (3.2)
    • C12Y302/01—Glycosidases, i.e. enzymes hydrolysing O- and S-glycosyl compounds (3.2.1)
    • C12Y302/01023—Beta-galactosidase (3.2.1.23), i.e. exo-(1-->4)-beta-D-galactanase

Definitions

  • Enzymes can be used to modify the flavor, texture and aroma of food and beverages.
  • One example of enzyme use in the food industry is found in cheese making.
  • Cheese manufacturers use enzymes to make cheese and enhance flavor.
  • rennet a protease
  • Other enzymes turn bland cheese curd into different flavored cheeses.
  • the flavors of most natural cheeses are due to enzymes produced by microflora naturally present in cheese, while the flavors of enzyme modified cheeses (EMCs) are due to enzymes added during the production process.
  • EMCs are a type of processed cheese produced by adding enzymes, such as lipase and/or protease, to immature cheese to impart desired flavor.
  • EMCs are typically made from young (immature) cheese (such as mild cheese curds) to which enzymes are added to develop the desired cheese flavor in a short time period ( e.g., in about 24 hours).
  • the flavor of an EMC can be 10 times as strong as a natural cheese, and depends largely on the enzyme reaction used to produce the EMC.
  • EMC is used as a processed cheese product, or as a powder that can be added to other food products to impart a cheese flavor, such as snack chips, soups, etc.
  • EMCs are commonly produced with lipases. Lipases break down the lipids present in the cheese, releasing fatty acids that impart flavor. For example, the release of high levels of butyric acid imparts "blue" flavor notes to cheese. Different lipases have different fatty acid profiles that result in different flavors.
  • EP 1 915 913 A1 describes methods for manufacturing flavored milk powder-like products by fermentation with microorganisms, amino acids and enzymes and products obtained by these methods with "honey-cream”, “cream-butter”, “cocoa-cream and malt-cream” and “caramel” flavor. However, EP 1 915 913 A1 does not disclose methods for enhancing cheese flavor.
  • a method of preparing a cheese food product comprising contacting a cheese food composition comprising lipids and lactose with one or more lipases and one or more lactases, wherein at least one of the one or more lactases exhibits galactose transferring activity as characterized in the claims.
  • at least one of the one or more lipases preferentially hydrolyzes short chain fatty acids before hydrolyzing long chain fatty acids.
  • the method further comprises adding lactose to the food composition.
  • the method further comprises an enzyme inactivation step.
  • the lipase comprises a lipase EC 3.1.1.3 produced by Rhizopus oryzae fermentation, a lipase EC 3.1.1.3 produced by Mucor javanicus fermentation, a lipase EC 3.1.1.3 produced by Aspergillus niger fermentation, a lipase EC 3.1.1.3 produced by Penicillium camemberti fermentation, and/or a lipase EC 3.1.1.3 produced by Penicillium roqueforti fermentation.
  • the lactase comprises ⁇ -galactosidase EC 3.2.1.23 produced by Bacillus circulans fermentation.
  • the cheese food product has a higher ratio of free short chain fatty acids to free long chain fatty acids than a comparable product treated with the one or more lipases but not the one or more lactases.
  • the cheese food product has stronger cheese flavor than a comparable product treated with the one or more lipases but not the one or more lactases.
  • the cheese food product has less of a soapy flavor than a comparable product treated with the one or more lipases but not the one or more lactases.
  • the cheese food product is selected from enzyme modified cheese and natural cheese. In some embodiments, the cheese food product is a final food product. In some embodiments, the cheese food product is a food additive.
  • a cheese food product prepared by a method comprising contacting a cheese food composition comprising lipids and lactose with one or more lipases and one or more lactases, wherein at least one of the one or more lactases exhibits galactose transferring activity.
  • the cheese food product has a higher ratio of free short chain fatty acids to free long chain fatty acids than a comparable product treated with the one or more lipases but not the one or more lactases.
  • the cheese food product has stronger cheese flavor than a comparable product treated with the one or more lipases but not the one or more lactases.
  • the cheese food product has less of a soapy flavor than a comparable product treated with the one or more lipases but not the one or more lactases.
  • the cheese food product is selected from enzyme modified cheese and natural cheese. In some embodiments, the cheese food product is a final food product. In some embodiments, the cheese food product is a food additive.
  • an enzyme composition comprising one or more lipases and one or more lactases, wherein at least one of the one or more lipases preferentially hydrolyzes short chain fatty acids before hydrolyzing long chain fatty acids, and at least one of the one or more lactases exhibits galactose transferring activity.
  • the enzyme composition further comprises a buffer.
  • the enzyme composition further comprises lactose.
  • short chain fatty acid means a fatty acid comprising 4-6 carbons.
  • long chain fatty acid means a fatty acid comprising 14-18 or more carbons.
  • medium chain fatty acid means a fatty acid comprising 8-12 fatty acids.
  • the enzymatic methods described herein provide methods of making cheese food products and methods of modifying cheese food flavor, such as EMC flavor, using, a lipase and a lactase.
  • Related enzyme compositions comprising, for example, a lipase and a lactase, and cheese food products produced by the described methods also are provided.
  • EMCs are commonly produced with lipases, which break down the lipids (including triglycerides) present in the cheese, releasing fatty acids that impart flavor.
  • the free fatty acid profile of a cheese affects the flavor of the cheese, and varies with the specific enzyme used.
  • Butyric acid a short chain fatty acid with four carbon atoms, C4
  • PGE pre-gastric enzyme
  • the enzymatic methods described herein use a lipase and a lactase to impart desired flavor to a cheese food product, such as to EMCs.
  • a typical lipase acts on a typical triglyceride (lipid) present in cheese
  • the terminal short chain (C4) fatty acid of the triglyceride is released first, followed by the terminal long chain fatty acid, with the middle fatty acid often not being released.
  • This reaction is illustrated in Figure 1 . It has been discovered that by using a lactase in conjunction with a lipase, release of short chain (C4) fatty acids can be achieved with reduced or eliminated release of long chain fatty acids.
  • Lactase hydrolyzes lactose into its component sugars, glucose and galactose. Most lactases also have a secondary activity of transferring galactose moieties to another lactose molecule, thus building oligosaccharides. While not wanting to be bound by any theory, it is believed that the use of lactase as described herein transfers a galactose moiety from lactose that is also present in the cheese food product (or that may be added to the cheese food product) to the site of the triglyceride from which the short chain fatty acid was released. Again, while not wanting to be bound by any theory, it is believed that the resulting molecule is not recognized by the lipase, and so is resistant to further hydrolysis that otherwise would have released long chain fatty acids. This reaction scheme is illustrated in Figure 2 .
  • Lipase is added to a food composition that comprises lipids and lactose, such as young cheese, cheese curds, or EMC base, and the lipase preferentially releases short chain (e.g., C4) fatty acids from the lipids, producing a desirable cheesy flavor, and leaving a free hydroxyl group on the lipid where the fatty acid was released.
  • short chain e.g., C4
  • Lactase is added to the food composition and transfers a galactose moiety (from lactose present in and/or added to the composition) to the free hydroxyl position on the lipid, resulting in a molecule that is resistant to further hydrolysis by the lipase.
  • lactose is added to the food composition to promote the galactose transfer reaction.
  • lactose may be present in a reaction buffer.
  • the enzymes can be added to the food composition at the same time or sequentially. In some embodiments, the enzymes are added substantially at the same time. In other embodiments, the enzymes are provided in a single composition that is added to the food composition.
  • the enzymes (and optional lactose) can be added to the food composition by any means, such as by mixing or blending the enzymes with the food composition, or by spraying the enzymes onto the food composition.
  • One or more lipases and one or more lactases are used.
  • the use of more than one lipase and more than one lactase may permit further control over the flavor or production process (e . g ., reaction conditions or reaction time).
  • different lipases and/or different lactases can be used in combination to achieve a desired enzyme activity profile (e.g., a desired lipase activity (including free fatty acid profile), a desired lactase activity, and/or a desired secondary lactase activity).
  • the amount of one or more of the enzymes is selected to permit further control over the flavor or production process.
  • the amount of lipase and/or ratio of two or more lipases may be selected to achieve a desired flavor.
  • the amount of lactase and/or ratio of two or more lactases may be selected to achieve a desired flavor. Indeed the selection of the type and amount of both the lipase(s) and lactase(s) may impact the free fatty acid profile and, thus, impact flavor.
  • the amount of enzyme used can be expressed in any known means, such as molar amounts or molar ratios (e . g ., nanomoles or micromoles of enzyme), weight amounts or weight ratio (micrograms or nanograms of enzyme), or activity amounts or activity ratios ( e . g ., "units" of enzyme or enzyme activity/weight or mole of enzyme). Standard methods of determining units of lipase and lactase activity are known in the art.
  • the methods use an amount of enzymes sufficient to increase the ratio of free small chain fatty acids to long chain fatty acids in the food product, relative to a comparable sample of the same cheese food product that has been treated with lipase but not with lactase.
  • the methods use an amount of enzymes sufficient to enhance the flavor of a cheese food product.
  • the exact amount of enzymes to be used will vary depending on the nature of the cheese food product, the desired flavor, and the concentration or activity of the enzymes. It should be understood that flavor includes but is not limited to the taste and aroma characteristics of the product. Enhanced flavor can be assessed by conventional means, such as by the use of professional or non-professional taste testers.
  • the methods provide a cheese food product with an enhanced cheesy flavor and/or a reduced soapy flavor.
  • the method also includes an inactivation step to inactivate the enzymes.
  • the method may include a heat inactivation step that comprises heating the enzyme-treated food composition for a time and at a temperature that is sufficient to inactivate one or more of the enzymes (or enzyme activities) present in the composition.
  • Suitable inactivation temperatures and times can be readily determined by those skilled in the art. Exemplary temperatures range from about 70°C to about 90°C. Exemplary times range from about 5 to about 60 minutes, including from about 5 to about 30 minutes.
  • the inactivation step comprises a pasteurization process, such as is conventional in the art for EMC products.
  • the inactivation step is selected such that enzyme activity is reduced or eliminated without sacrificing the quality of the cheese food product.
  • an enzyme inactivation step is not required to prevent development of undesirably soapy flavors that otherwise might arise from the release of long chain fatty acids.
  • the methods do not include an enzyme inactivation step.
  • the enzymatic methods described herein can use any enzymes that are safe for use in food production.
  • the enzymes can be obtained from any source, and can be derived from any source, including animal or microbial.
  • the enzymes can be obtained from microorganisms that produce the enzymes naturally or that have been genetically modified to produce one or more enzymes, using methods well known in the art.
  • Enzymes also can be obtained by recombinant methods, such as from transformed or transfected cells, by methods well known in the art.
  • a nucleic acid sequence encoding a desired enzyme can be inserted into an expression vector, which can be used to transform or transfect a host cell for production of the enzyme.
  • Many suitable enzymes are commercially available, as discussed below.
  • Lipases are a class of hydrolases that act to hydrolyze the ester bonds of lipid substrates, such as triglycerides.
  • Triglycerides comprise a glycerol molecule esterified with three fatty acids ( see e.g ., Figures 1 and 2 ).
  • the fatty acids of a triglyceride may be short chain (e.g., comprising 4-6 carbons) or may be medium or long chain (e.g., comprising 8-12 or more carbons). Fatty acid chain lengths of 16, 18, and 20 carbons are the most common in naturally occurring triglycerides.
  • the fatty acids present on a single triglyceride may be the same or different.
  • triglycerides present in dairy products typically include a short chain fatty acid such as butyric acid (C4), and two longer chain fatty acids, such as palmitic acid (C16) and stearic acid (C18).
  • the lipase preferentially hydrolyzes the short chain fatty acids first, before hydrolyzing longer chain fatty acids.
  • preferentially hydrolyzes the short chain fatty acids first means that the lipase preferentially releases the short chain fatty acid from a lipid before releasing long chain fatty acids, such that the lipase is more likely to release the short chain fatty acid first.
  • Figure 1 illustrates a lipase systematically hydrolyzing the short chain fatty acid (at position 3 in the figure) of a triglyceride followed by hydrolysis of the longer chain fatty acid (at position 1 in the figure).
  • This desired preferential activity can be confirmed by adding the lipase to a composition comprising triglycerides comprising long chain and short chain fatty acids, stopping the lipase reaction early in the process, before the lipids are completely hydrolyzed, and analyzing the free fatty acid profile (or the remaining partially hydrolyzed triglyceride) to confirm that short chain fatty acids were preferentially released, e . g ., that more short chain fatty acids than long chain fatty acids had been released at the time the reaction was stopped.
  • Such an analysis can be undertaken by methods known in the art.
  • CHEESEMAX ® One exemplary lipase that can be used in the methods described herein is sold under the name CHEESEMAX ® (Amano Enzyme, U.S., Elgin, IL).
  • CHEESEMAX ® is sold as a preparation with a lipase activity of not less than 7,500 U/g as assessed by the Food Chemical Codex IV method; one unit is the amount of enzyme that releases 1 ⁇ mole of butyric acid in one minute at pH 7.0).
  • CHEESEMAX ® is a food grade lipase preparation produced by Rhizopus oryzae fermentation under Good Manufacturing Practices.
  • CHEESEMAX ® has a molecular weight of 38,000 and an isoelectric point of 6.8. CHEESEMAX ® can be inactivated by heating at 80°C for about 15 minutes. Some of the characteristics of CHEESEMAX ® (temperature and activity, pH and activity, thermostability and pH stability) are shown in Figures 4-7 . CHEESEMAX ® hydrolyzes triglyceride short, medium and long-chain fatty acids with a preference for short and medium chain fatty acids at the 1 and 3 positions of triacylglycerides. Commercial preparations of CHEESEMAX ® may be used at the concentration provided, or can be diluted or further concentrated for use. Other lipases produced by Rhizopus oryzae fermentation also can be used as described herein.
  • Lipase M is a food grade lipolytic enzyme preparation produced by Mucor javanicus fermentation under Good Manufacturing Practices.
  • Lipase M can hydrolyze short, medium and long chain fatty acids at 1, 2, and 3 positions of tri- di- and monoglycerides. Lipase M is sold at not less than 10,000 units/gram lipase activity. Commercial preparations of Lipase M may be used at the concentration provided, or can be diluted or further concentrated for use. Some of the characteristics of Lipase M (temperature and activity, pH and activity, thermostability and pH stability) are shown in Figures 8A-8D . Other lipases produced by Mucor javanicus fermentation also can be used as described herein.
  • Lipase A 12 is a food grade triacylglycerol lipase preparation produced by Aspergillus niger fermentation under Good Manufacturing Practices. Lipase A12 can hydrolyze short, medium and long-chain fatty acids at 1, 2, and 3 positions of tri-, di- and monoglycerides. Lipase A12 has a molecular weight of 35,000 and an isoelectric point of 4.10. Lipase A12 is sold at not less than 120,000 units/gram lipase activity.
  • Lipase A12 Commercial preparations of Lipase A12 may be used at the concentration provided, or can be diluted or further concentrated for use. Additional characteristics of lipase A12 are shown in Figures 9A-D . Other lipases produced by Aspergillus niger fermentation also can be used as described herein.
  • lipase DF15 Another exemplary lipase that can be used with the methods described herein is sold under the name Lipase DF "Amano" 15-K (hereinafter “lipase DF15”) (Amano Enzyme, U.S., Elgin, IL).
  • Lipase DF15 is produced by Rhizopus oryzae fermentation. This food-grade lipase product has a positional specificity for the 1- and 3-positions of glycerides, and hydrolyzes ester bonds of 1( ⁇ )- and 3( ⁇ )- positions of triglycerides.
  • Lipase DF15 is relatively specific to fatty acids with long and medium chain length.
  • Lipase activity (by the Food Chemical Codex V method at pH 7) is not less than 150,000 units/gram.
  • Commercial preparations of Lipase DF15 may be used at the concentration provided, or can be diluted or further concentrated for use. Additional characteristics of lipase DF15 are shown in Figures 10A-D .
  • Other lipases produced by Rhizopus oryzae fermentation also can be used as described herein.
  • Lipase R is a food grade triacylglycerol lipase produced by Penicillium roqueforti fermentation under Good Manufacturing Practices. Lipase R hydrolyzes short-chain and medium-chain fatty acids in preference to long-chain fatty acids from 1 and 3 positions of tri-, di- and monoglycerides.
  • Lipase R has a molecular weight of 25,000, an isoelectric point of 4.50, and inactivation conditions (0.1% enzyme solution) of 60°C for 2 minutes or 70°C for 1 minute. Lipase R is sold at not less than 900 units/gram lipase activity. Commercial preparations of Lipase R may be used at the concentration provided, or can be diluted or further concentrated for use. Additional characteristics of lipase R are shown in Figures 11A-D . Other lipases produced by Penicillium roqueforti fermentation also can be used as described herein.
  • Lipase G50 is a food grade enzyme preparation produced by Penicillium camembertii fermentation under Good Manufacturing Practices. Lipase G50 has high esterifying activity and hydrolyzes glycerides, and partial glycerides more rapidly than triglyceride, producing glycerol and fatty acid. Lipase G50R is sold at not more than 50,000 units/gram lipase activity.
  • Lipase G50 Commercial preparations of Lipase G50 may be used at the concentration provided, or can be diluted or further concentrated for use. Additional characteristics of lipase G50 are shown in Figures 12A-D . Other lipases produced by Penicillium camembertii fermentation also can be used as described herein.
  • Lactases are another class of hydrolases that hydrolyze the disaccharide lactose into its component monomers, glucose and galactose. As noted above, most lactases also have a secondary activity of transferring galactose moieties to another lactose molecule, and do so repeatedly, thus building oligosaccharides.
  • lactases are added to dairy products to reduce lactose content to make the products more acceptable for people suffering from lactose-intolerance.
  • lactases from Bacillus circulans, Kluyveromyces fragilis, Kluyveromyces lactis and Aspergillus oryzae are commercially available. Any of these or other lactases with the desired secondary activity can be used in the methods described herein.
  • the desired secondary activity can be confirmed by adding the lactase to a composition comprising lactose and analyzing the resulting oligosaccharide content to confirm that the lactase transferred galactose moieties onto lactose molecules to build oligosaccharides.
  • enzymes are selected or engineered to have a high level of the secondary (galactose-transferring).
  • a lactase isolated from Aspergillus oryza has been engineered to have high levels of this activity, and is sold under the name BIOLACTATM (Amano Enzyme, U.S., Elgin, IL).
  • BIOLACTATM is a neutral lactase ( ⁇ -galactosidase, Nomenclature Committee of the International Union of Biochemistry and Molecular Biology (NC-IUBMB) number: EC3.2.1.23) produced by the controlled fermentation of Bacillus circulans.
  • BIOLACTATM has an optimum pH of about 6.0 ( Figure 13B ), is stable at between about pH 5.0-9.5 ( Figure 13D ), has an optimum temperature of about 65°C ( Figure 13A ), and is stable for at least an hour at about 55°C ( Figure 13C ).
  • the working temperature of BIOLACTATM is practically applicable enough at 60° C in the presence of 50% lactose ( Figure 14A ).
  • the hydrolytic speed of BIOLACTATM e.g ., hydrolysis of lactose in fresh milk
  • BIOLACTATM is directly proportional to the amount of BIOLACTATM added ( Figure 14B ).
  • BIOLACTATM In the presence of 55% lactose, BIOLACTATM produces oligosaccharide of about 58% including ditri-, tetra- and penta- saccharide ( Figure 14C ).
  • One lactose unit (LU) is defined as the amount of enzyme that liberates 1 ⁇ mol of glucose per minute at the early stage of the reaction at 40°C and pH 6.0.
  • BIOLACTATM is suitable for use in the methods described herein. BIOLACTATM is sold in commercial preparation with a lactase activity of 5,500 LU/g.
  • the lipase used is CHEESEMAX® and the lactase used is BIOLACTATM.
  • BIOLACTATM CHEESEMAX® and the lactase used.
  • CHEESEMAX® and the lactase used is BIOLACTATM.
  • BIOLACTATM about 0.1-0.2% CHEESEMAX ® and about 0.1-0.2% BIOLACTATM can be used.
  • these amounts are exemplary only, and those skilled in the art will recognize that different amounts of different enzymes may be used, depending on enzyme activity and desired affect.
  • the lipase comprises a lipase produced by Rhizopus oryzae fermentation, a lipase produced by Mucor javanicus fermentation, a lipase produced by Aspergillus niger fermentation, a lipase produced by Rhizopus oryzae fermentation, a lipase produced by Penicillium camemberti fermentation, and/or a lipase produced by Penicillium roqueforti fermentation, and the lactase comprises ⁇ -galactosidase EC3.2.1.23 produced by Bacillus circulans fermentation.
  • the lipase comprises CHEESEMAX®, Lipase M, Lipase A12, Lipase D15, Lipase G50 and/or Lipase R, and the lactase comprises BIOLACTATM.
  • the lipase comprises a lipase produced by Mucor javanicus fermentation and the lactase comprises ⁇ -galactosidase EC 3.2.1.23 produced by Bacillus circulans fermentation, such as where the lipase comprises Lipase M the lactase comprises BIOLACTATM.
  • other lactases with galactose-transferring activity can be used in the methods described herein, such as in addition to or instead of BIOLACTATM in any of the described lipase/lactase combinations.
  • lipase(s) and lactase(s) can be screened for a desired effect on EMC as illustrated in the examples below.
  • a given combination may have a desired effect on one or more of free fatty acid content, cheesiness, sharpness, lack of soapiness, and aroma.
  • lactose is used to promote the transfer of galactose moieties from lactose molecules to the partially hydrolyzed lipids.
  • Lactose is available commercially from a number of sources, including Sigma Chemical Co.
  • the enzymes can be provided in compositions typically used for the purpose of EMC manufacture, which may include, for example, one or more buffers to control pH.
  • one or more buffers can be used to control the pH to that range.
  • Exemplary buffers include but are not limited to acetate buffer and phosphate buffer.
  • compositions comprising one or more lipases and one or more lactases, wherein at least one of the one or more lipases preferentially hydrolyzes short chain fatty acids before hydrolyzing long chain fatty acids, and at least one of the one or more lactases exhibits galactose transferring activity.
  • the composition further comprises a buffer.
  • cheese food products that have been prepared using the enzymatic methods described herein.
  • “food products” includes food and beverage products at all stages of production.
  • the cheese food products is a dairy product.
  • Dairy products include but are not limited to cheese, cheese curds, cream cheese, cottage cheese and the like. Dairy products include foods and beverages that are final products and/or that are used as a component of a final product.
  • the cheese food products include EMC prepared using the enzymatic methods described herein.
  • the cheese food products include traditional cheese (e.g., hard cheese) prepared using the enzymatic methods described herein.
  • the cheese food product is used to impart a dairy flavor to other products, such as a food additive.
  • EMC prepared by the methods described herein can be used to impart a cheesy flavor to snack foods, soups, breads, etc.
  • the cheese food products described herein e.g., prepared by the enzymatic methods described herein, have a free fatty acid content that is different from a comparable cheese food product that has not been prepared by the enzymatic methods described herein, e.g. , that has been prepared with lipase only, and not with a lactase that has a secondary (galactose-transferring) activity.
  • Free fatty acid content can be assessed by methods known in the art. Typical methods include extraction of fatty acids from the cheese food product, conversion to methyl esters, and analysis by gas chromatography, as illustrated in the examples below.
  • the cheese food product has a higher ratio of free short chain fatty acids to free long chain fatty acids than a comparable product treated with one or more lipases but not treated with one or more lactases having galactose transferring activity.
  • the cheese food product has a long chain fatty acid content, such as a C18 fatty acid content, that is about 2/3 or less (e .g ., 66% or less, including 63%) than the long chain fatty acid content of a comparable product treated with one or more lipases but not treated with one or more lactases having galactose transferring activity.
  • the cheese food product has a short chain fatty acid content, such as a C4 fatty acid content, that is about 90% or more (including 91 %) of the short chain fatty acid content of a comparable product treated with one or more lipases but not treated with one or more lactases having galactose transferring activity.
  • a short chain fatty acid content such as a C4 fatty acid content
  • the cheese food product has stronger cheese flavor than a comparable product treated with one or more lipases but not one or more lactases having galactose transferring activity. In some embodiments, the cheese food product has less of a soapy flavor than a comparable product treated with one or more lipases but not one or more lactases having galactose transferring activity.
  • Examples 1-4 demonstrate that methods described herein result in improved cheese flavor and reduced soapy flavor of EMC. Similar methods can be used to modify the flavor of tradition cheese, but a longer flavor development time would be required for a solid cheese composition (e . g ., cheddar cheese) as compared to the liquefied cheese composition used below.
  • a solid cheese composition e . g ., cheddar cheese
  • Weyauwega Star Dairy Cheddar Cheese Curd was purchased from a local grocery store. About 75 grams (g) of the Cheddar Cheese Curd was weighed into a Cusinart. 75 milliliters (ml) of buffer containing lactose (0.5% w/v lactose, 1.0% w/v NaCl, and 1.5% w/v sodium citrate) was gradually added as the curds were processed into a slurry. Two 50 gram aliquots of the slurry were weighed into sterile polycarbonate flasks, and the samples were then pasteurized for 30 minutes in boiling water. The samples were allowed to cool for 1 hour at 50° C.
  • the samples were each tasted twice. For the first tasting, 2.0 g of the solid part of the samples was weighed into a weighing dish, and the weight was brought to 10.0 g with EASY CHEESE ® American Pasteurized Cheese Snack. The sample was mixed thoroughly with a spoon before a single taster tried the sample. Each sample was compared to a control (the EASY CHEESE ® alone) and rated on cheesiness and soapiness. Thus, the first taste test focused on qualitative differences. Results are shown below in Table 1.
  • Table 1 Results of Taste Test #1 Sample Description of Taste Control cheese snack Bland, not very cheesy CHEESEMAX ® treated EMC Increased cheesiness (more than other two samples), strong soapiness CHEESEMAX ® + BIOLACTATM treated EMC More cheesy than control (but not as much as CHEESEMAX ® only sample), not as soapy as CHEESEMAX ® only sample
  • each EMC prepared as described above was weighed into a 50 ml centrifuge tube. The following reagents were added to each tube: 1 ml 2.5 M H 2 SO 4 , 3 ml water, and 5 ml internal standard (C5 (valeric acid, purchased from Aldrich, catalog number 240370) and C17 (heptadecanoid acid, purchased from Sigma, catalog number H3500), 1 mg/ml of each fatty acid in 1:1 ether:hexane). Since the EMC was too sticky to emulsify when shaken by hand, each sample was homogenized for one minute with a Polytron PT 1200 E handheld homogenizer at maximum RPM.
  • C5 valeric acid, purchased from Aldrich, catalog number 240370
  • C17 heptadecanoid acid
  • the samples were centrifuged for 10 minutes at 3000 RPM in a Beckman Coulter AllegraTM 25R Centrifuge, System ID 433500, and then centrifuged again for 20 minutes at 3500 RPM to obtain better separation of layers.
  • the oil layers were drawn off with a pipette and allowed to pass through SEP columns equilibrated with 10 ml heptane. The columns were washed with 10 ml 2:1 chloroform:propanol, and the free fatty acids (FFA) were eluted with 5 ml 2% formic acid in ether.
  • FFA free fatty acids
  • Samples were run on a gas chromatograph system, Model 6890, manufactured by Aglient Technologies, with a split/splitless inlet, a split liner, and a pulsed split inlet model.
  • the split ratio was 50:1 and the split flow 109 ml/min.
  • the inlet temperature was 250°C, and the head pressure was 230 kPa.
  • the column used was 0.15 um DB-23, 60 m x 0.25 mm ID.
  • the total gas flow was 113 ml/min, and the carrier gas was helium. Helium flow was 2.2 ml/min, helium make-up flow was 30 ml/min, hydrogen flow was 40 ml/min, and air was 800 ml/min.
  • the average velocity was 34 cm/sec.
  • the oven was programmed as shown below in Table 3, with the detector temperature set at 280°C: Table 3: Oven Program for FAME analysis Temperature (°C) Rate (°C/min) Final Temperature (°C) Hold Time (minutes) 50 N/A 50 4 50 25 175 0 175 4 230 0
  • the sample volume used was 1 ⁇ l. Each sample was injected five times, cycling through all three samples (i.e ., A, B, C, instead of A, A, A) so as not to bias the results by allowing one sample to sit untouched longer than the others. After each sample was run, the areas for each FAME peak (retention times previously determined) were entered into an Excel spreadsheet. Using the internal standards for conversion factors, the concentration of each free fatty acid in mmol/kg was calculated. Results of the gas chromatography analysis are shown below in Table 4 and Figure 3 . The average concentration of each fatty acid is represented in mmol/kg. Table 4: Average concentration of fatty acid for each EMC (mmol/kg) Fatty Acid FA Conc.
  • the CHEESEMAX ® treated EMC had a measured C4 concentration of 8.19 mmol and the CHEESEMAX ® + BIOLACTATM treated EMC had a measured C4 concentration of 7.43 mmol C4/kg.
  • the true concentrations of C4 in these samples may be as high as 11.47 mmol/kg and 10.40 mmol/kg, respectively, based on previous studies of this phenomenon.
  • EMC was made with five different lipases: Lipase A12, Lipase DF15, Lipase G50, Lipase M, and Lipase R.
  • EMC was produced using the lipase both by itself and in conjunction with BIOLACTATM. After the EMC was produced, the free fatty acids were extracted, converted to methyl esters, and analyzed by gas chromatography. Samples of EMC were also tasted and rated on their cheesiness, sharpness, and soapiness.
  • each lipase was used with a different batch of EMC.
  • EMC EMC-Ethyl-Coupled Chemical Company
  • about 175 grams of the cheddar cheese curd was weighed into a Cusinart.
  • An equal volume of buffer (0.5% w/v lactose, 1.0% w/v NaCl, and 1.5% w/v sodium citrate) was gradually added as the curds were processed into a slurry.
  • Six 50g aliquots of the slurry were weighed into sterile polycarbonate flasks, and the samples were then pasteurized for 30 minutes in boiling water. The samples were allowed to cool for at least 1 hour at the optimum working temperature of the lipase being used.
  • the temperature used for each lipase is shown below in Table 5.
  • Table 5 Optimum working temperatures of lipases Lipase Temperature (°C) Lipase A12 50 Lipase DF15 40 Lipase G50 40 Lipase M 40 Lipa
  • each of one EMC sample treated only with the lipase and one EMC sample treated with both the lipase and BIOLACTATM was mixed with 8 g of pasteurized cheese spread.
  • the pasteurized cheese spread was also tasted by itself to establish base levels of flavors.
  • Each sample was rated from 0-10 (with 10 being the highest score possible) by a single taster on cheesiness, sharpness, and lack of soapiness.
  • bitterness was also detected in the EMC samples made with Lipase M.
  • the sample prepared with Lipase M only had a bitterness level of 4 (or 6 for lack of bitterness), while the sample prepared with both Lipase M and BIOLACTATM had a bitterness level of 2 (or 8 for lack of bitterness). All of the other EMCs would have bitterness levels of 0 (or 10 for lack of bitterness).
  • each EMC was weighed into a 50 ml centrifuge tube.
  • the following reagents were added to each tube: 1 ml 2.5 M H 2 SO 4 , 3 ml water, and 5 ml internal standard (C5 and C17, 1 mg/ml of each fatty acid in 1:1 ether:hexane).
  • the samples were vigorously vortexed to create emulsions.
  • the samples were centrifuged for 15-30 minutes at 3000 RPM in a Beckman Coulter AllegraTM 25R Centrifuge, System ID 433500.
  • the oil layers were drawn off with a pipette and allowed to pass through SEP columns equilibrated with 10 ml heptane.
  • the samples were run on a gas chromatograph system, Model 6890, manufactured by Aglient Technologies, with a split/splitless inlet, a split liner, and a pulsed split inlet model.
  • the split ratio was 50:1 and the split flow 109 ml/min.
  • the inlet temperature was 250°C, and the head pressure was 230 kPa.
  • the column used was 0.15 um DB-23, 60 m x 0.25 mm ID.
  • the total gas flow was 113 ml/min, and the carrier gas was helium. Helium flow was 2.2ml/min, helium make-up flow was 30 ml/min, hydrogen flow was 40 ml/min, and air was 800 ml/min.
  • the average velocity was 34 cm/sec.
  • the oven was programmed as shown below in Table 7, with the detector temperature set at 280°C.
  • Table 7 Oven Program for FAME Analysis Temperature (°C) Rate (°C/min) Final Temperature (°C) Hold Time (minutes) 50 N/A 50 4 50 25 175 0 175 4 230 0
  • the sample volume used was 1 ⁇ l. Each sample was injected three times, cycling through all six samples (i.e., A, B, C, instead of A, A, A) so as not to bias the results by allowing one sample to sit untouched longer than the others.
  • the concentration of each free fatty acid in mmol/kg was calculated using the internal standards for conversion factors,.
  • Table 8 shows the results of the gas chromatography analysis. Concentrations of free fatty acids that contribute to cheesy flavor (C4) or soapy flavor (C 14, C16, and C18) in the EMCs produced with the various lipases are shown: TABLE 8: Average Free Fatty Acid Concentration Levels in EMCs Made with Various Lipases Sample Average C4 Conc. (mmol/kg) Average C14 Conc. (mmol/kg) Average C16 Conc. (mmol/kg) Average C18 Conc.
  • Lipase A12 did not improve the taste of EMC relative to the control; however, both Lipase G50 and Lipase R produced improvements in cheesiness and sharpness.
  • the determined free fatty acid concentrations are not entirely consistent with the taste test results. It is possible that side reactions are affecting the determined free fatty acid concentrations or taste test results, or both.
  • the taste test results reported above were based on a single tasting by a single person, and so additional tasting by other people might produce results more consistent with the free fatty acid concentrations.
  • Lipase DF15 and Lipase M Two of the lipases, Lipase DF15 and Lipase M, produced C4 concentrations that are higher than those found in CHEESEMAX ® EMC. However, in taste tests, those EMCs (both lipase EMC and lipase/BIOLACTATM EMC) scored very low on cheesiness and sharpness, being comparable to or lower than the control cheese spread. Both EMCs made with Lipase DF 15 scored lowest on lack of soapiness (for high soapiness), so it is possible that soapiness may have masked any cheesy flavor present in the sample. The Lipase M EMCs also scored low on lack of soapiness, though not as low as the Lipase DF15 EMCs. Thus, for these enzymes as well, the determined free fatty acid concentrations are not entirely consistent with the taste test results.
  • Table 9 shows the ratios of long chain fatty acid in lipase+ BIOLACTATM EMC to the long chain fatty acid concentrations in the corresponding lipase EMC. For comparison, the ratios for CHEESEMAX ® are also presented. A ratio of less than one indicates that the use of the lipase/lactase combination reduced the long chain fatty acid content as compared to use of the lipase alone.
  • the ratio was about the same for each free fatty acid (C14, C16, C18). However, none of the lipases produced a ratio as low as that produced by CHEESEMAX ® (average ratio 0.66).
  • adding BIOLACTATM to the EMC produced more than twice the long chain fatty acid (ratio 2.14), even though the Lipase A12/BIOLACTATM EMC tasted less soapy than the Lipase A12 EMC.
  • Combining BIOLACTATM with Lipase DF15 produced less long chain fatty acid (average ratio 0.84) but slightly more soapy flavor during the taste test.
  • the EMC made with Lipase G50/BIOLACTATM had slightly higher long chain fatty acid concentrations (average ratio 1.17) and a slightly more soapy taste.
  • adding BIOLACTATM to the EMC reduced concentrations of all free fatty acid slightly (average ratio 0.95), but C4 was affected more strongly than the long chain fatty acid.
  • the Lipase M/BIOLACTATM EMC had slightly less soapy flavor than the Lipase M EMC.
  • the C4 concentrations were slightly higher in the Lipase R/BIOLACTATM EMC, but the long chain fatty acid concentrations were increased to a greater degree (average ratio 1.79).
  • the Lipase R/BIOLACTATM EMC scored better in all categories during the taste test than the Lipase R EMC. Thus, for three of the lipases, taste test results were not consistent with the gas chromatography results.
  • Lipase A12 Lipase G50
  • Lipase R Lipase R
  • Two lipases, Lipase DF15 and Lipase M produced lower long chain fatty acid concentrations when combined with BIOLACTATM.

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Claims (15)

  1. Verfahren zur Herstellung eines Käse-Nahrungsmittelprodukts umfassend In-Kontakt-Bringen einer Käse-Nahrungsmittelzusammensetzung umfassend Fette und Laktose mit einer oder mehreren Lipasen und einer oder mehreren Laktasen, wobei mindestens eine der einen oder mehreren Laktasen Galaktose-transferierende Aktivität aufweist, wobei das Käse-Nahrungsmittelprodukt einen stärkeren Käsegeschmack hat und ein höheres Verhältnis von freien kurzkettigen Fettsäuren zu freien langkettigen Fettsäuren als ein vergleichbares Produkt, behandelt mit der einen oder mehreren Lipasen aber nicht der einen oder mehreren Laktasen.
  2. Verfahren nach Anspruch 1, wobei mindestens eine der einen oder mehreren Lipasen bevorzugt kurzkettige Fettsäuren hydrolysiert bevor sie langkettige Fettsäuren hydrolysiert.
  3. Verfahren nach einem der Ansprüche 1 und 2, weiter umfassend die Zugabe von Laktose zu der Nahrungsmittelzusammensetzung.
  4. Verfahren nach einem der Ansprüche 1-3, weiter umfassend einen Enzym-Inaktivierungsschritt.
  5. Verfahren nach einem der Ansprüche 1-4, wobei das Käse-Nahrungsmittelprodukt weniger von einem seifigen Geschmack hat als ein vergleichbares Produkt, behandelt mit der einen oder mehreren Lipasen aber nicht der einen oder mehreren Laktasen.
  6. Verfahren nach einem der Ansprüche 1-5, wobei das Käse-Nahrungsmittelprodukt ausgewählt ist aus enzymmodifiziertem Käse und Naturkäse.
  7. Verfahren nach einem der Ansprüche 1-6, wobei das Käse-Nahrungsmittelprodukt ein Nahrungsmittelendprodukt ist.
  8. Verfahren nach einem der Ansprüche 1-7, wobei das Käse-Nahrungsmittelprodukt ein Lebensmittelzusatzstoff ist.
  9. Verfahren nach einem der Ansprüche 1-8, wobei die Lipase eine Lipase umfasst, ausgewählt aus der Gruppe bestehend aus einer Lipase EC 3.1.1.3 gewonnen aus Aspergillus niger Fermentation, einer Lipase EC 3.1.1.3 gewonnen aus Rhizopus oryzae Fermentation, einer Lipase EC 3.1.1.3 gewonnen aus Penicillium camemberti Fermentation, einer Lipase EC 3.1.1.3 gewonnen aus Mucor javanicus Fermentation, und einer Lipase EC 3.1.1.3 gewonnen aus Penicillium roqueforti Fermentation, und wobei die Laktase β-Galaktosidase EC 3.2.1.23 umfasst, gewonnen aus Bacillus circulans Fermentation.
  10. Käse-Nahrungsmittelprodukt hergestellt durch ein Verfahren nach einem der Ansprüche 1-9, wobei das Käse-Nahrungsmittelprodukt ausgewählt ist aus enzymmodifiziertem Käse und Naturkäse.
  11. Käse-Nahrungsmittelprodukt nach Anspruch 10, wobei das Nahrungsmittelprodukt weniger von einem seifigen Geschmack hat als ein vergleichbares Produkt, behandelt mit der einen oder mehreren Lipasen aber nicht der einen oder mehreren Laktasen.
  12. Käse-Nahrungsmittelprodukt nach einem der Ansprüche 10-11, wobei das Käse-Nahrungsmittelprodukt ein Nahrungsmittelendprodukt ist.
  13. Käse-Nahrungsmittelprodukt nach einem der Ansprüche 10-12, wobei das Käse-Nahrungsmittelprodukt ein Lebensmittelzusatzstoff ist.
  14. Enzymzusammensetzung umfassend eine oder mehrere Lipasen und eine oder mehrere Laktasen, wobei mindestens eine der einen oder mehreren Lipasen bevorzugt kurzkettige Fettsäuren hydrolysiert bevor sie langkettige Fettsäuren hydrolysiert, und mindestens eine der einen oder mehreren Laktasen Galaktose-transferierende Aktivität aufweist.
  15. Enzymzusammensetzung nach Anspruch 14, weiter umfassend Laktose.
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US20230248019A1 (en) 2020-07-13 2023-08-10 Amano Enzyme Inc. Method for producing plant protein food
US20220022478A1 (en) 2020-07-27 2022-01-27 Sargento Foods Inc. Flavor Ferment to Produce Natural Cheese With Specific Flavor Attributes
US11510416B1 (en) 2021-02-18 2022-11-29 Sargento Foods Inc. Natural pasta-filata style cheese with improved texture
WO2024166995A1 (ja) * 2023-02-10 2024-08-15 天野エンザイム株式会社 加工脂肪酸含有組成物の製造方法及びこれらの応用

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JP2005517417A (ja) * 2002-02-21 2005-06-16 フォンテラ コ−オペレイティブ グループ リミティド 風味増強エステルの製造方法
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CA2594953A1 (en) 2005-01-17 2006-07-27 Novozymes North America, Inc. Methods for flavor enhancement
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